Molecular Cloning and Expression Analysis of CML27 in Brassica oleracea Pollination Process

Abstract

To keep genetic diversity, flowering plants have developed a self-incompatibility system, which can prevent self-pollination. It has been reported that calcium concentration in pistil papilla cells was increased after self-pollination in transformed self-incompatible Arabidopsis thaliana. In this study, we found that CML27 changed its expression level for both mRNA and protein when compared to transcriptome and proteome. At the same time, CML27 was expressed in the anther and pistil at a high level and reached up to 5-fold up-regulated expression in the pistil at 1 h post-pollination when compared to 0 min. In order to find out potential proteins that may interact with BoCML27, BoCML27 was expressed in and isolated from E. coli. After its co-incubation with Brassica oleracea pistil proteins, the products were separated on SDS-PAGE gels. We found a specific band at the position between 130–180 kDa. Through LC-MS-MS (Q-TOF) analysis, eight proteins were identified from the band. The proteins include 26S proteasome non-ATPase regulatory (26S), Phospholipase D, alpha 2 (PLDα2) involved in Ca2+ binding and Coatomer subunit alpha-2-like (Coatomer) involved in vesicle mediated transport. All of these identified proteins provide new insights for the self-incompatibility response in B. oleracea, specific for increasing Ca2+ concentration in pistil papilla cells.